We have demonstrated that growing E. coli in the presence of 5-azacytidine causes a decrease in activity of the DNA methylase assayed in vitro. This decrease is not due to the inhibitory affect of 5-azacytidine on protein or RNA synthesis since neither rifampin nor chloramphenicol causes a decrease in this activity. The decrease is due to a loss of activity of the DNA(cytosine-5)methyltransferase, the DNA(adenine-N6)methyltransferase is not affected. The affect is not readily reversible by dialyzing extracts nor have we been able to inhibit the enzyme in vitro with 5-azacytidine or extract of cells treated with 5-azacytidine. Preliminary data indicate that DNA extracts of cells treated with 5-azacytidine inhibit the DNA-methylase in vitro. In this proposal we will investigate the basis for this inhibition. We will determine if DNA synthesis in the presence of 5-azacytidine is necessary for inhibition to occur and if there is an actual decrease in in the amount of 5-methylcytosine in DNA in the bacterial cell. Experiments will be performed to determine if the inhibition of DNA methylase and inhibition of growth caused by 5-azacytidine are due to some common intermediate which leads to these two effects. These experiments will be performed by determining if, in mutants deficient in the ability to deaminate or phosphorylate 5-azacytidine, both effects of the drug decrease in parallel. The ability of pyrimidine nucleosides to reverse these two effects of the drug will also be studied. Since we have found that DNA extracted from cells grown in the presence of 5-azacytidine inhibits the enzyme in viiro we will determine whether the 5-azacytidine must appear in the site normally methylated by the E. coli K12 DNA(cytosine-5)methyltransferase for inhibition to occur. Since inhibition of the enzyme activity appears to be only slowly reversible we will demonstrate whether or not this is due to covalent attachment of the inhibitor to the enzyme, and the nature of this attachment.